Bottom Line:
Herein, we present the initial results of our studies aimed at the design of bifunctional peptide-based compounds.The chemical approach, that was utilized in this work, was based on the conjugation of antimicrobial peptides with the peptides, that have potential pro-proliferative and/or cytoprotective activity towards human keratinocytes and fibroblasts, in order to obtain antimicrobials with reduced cytotoxicity or compounds that maintain both activities, i.e. inhibit bacterial or fungi growth and activate cell proliferation/migration in in vitro tests.As a result, we obtained a group of peptide conjugates that effectively inhibited the growth of selected bacterial and fungi strains and were able to stimulate proliferation and migration of keratinocytes and fibroblasts under their effective microbicidal concentrations.

Affiliation: Department of Biochemistry, Faculty of Chemistry, University of Gdansk, Gdansk, Poland.

ABSTRACTSkin represents the largest organ of the human body and plays a crucial role in its protection from the negative impact of the outside environment, maintains its homeostasis, enables sensory interaction and thermoregulation. The traumatized skin tissue undergoes several phenotype switches due to progressive reoxygenation and release of cytokine and growth factors, that activate mechanisms of reparative processes. However, in case of wounds colonized with pathogenic microflora natural regenerative mechanisms become substantially impaired, that could lead to chronic inflammatory states with non-healing skin lesions. Herein, we present the initial results of our studies aimed at the design of bifunctional peptide-based compounds. The chemical approach, that was utilized in this work, was based on the conjugation of antimicrobial peptides with the peptides, that have potential pro-proliferative and/or cytoprotective activity towards human keratinocytes and fibroblasts, in order to obtain antimicrobials with reduced cytotoxicity or compounds that maintain both activities, i.e. inhibit bacterial or fungi growth and activate cell proliferation/migration in in vitro tests. As a result, we obtained a group of peptide conjugates that effectively inhibited the growth of selected bacterial and fungi strains and were able to stimulate proliferation and migration of keratinocytes and fibroblasts under their effective microbicidal concentrations.

pone.0140377.g002: Effect of the peptides on HaCaT keratinocytes and fibroblasts cell migration in the in vitro scratch test.HaCaT cells and fibroblasts were grown in 6-well plates until the 100% confluence was reached, then cells were starved for the next 12 hours in DMEM without 10% FCS and scratched once vertically with a 200μL pipette tip. After being washed two times with sterile PBS, a fresh portion of DMEM medium was added and cells were treated with the tested peptides applied at their optimal doses (25 μg/mL). Migration was analyzed after 24 hours by means of Zeiss Observer D1 microscope, wounding areas were analyzed with AxioVision software and expressed as the percentage of the wound width in comparison to the control sample (cells incubated in DMEM without FCS). Control+ FCS corresponds to the sample with cells incubated in the medium containing 10% FCS and was treated as the additional positive control. Data represent the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control.

Mentions:
As illustrated in Fig 2 all of the tested peptides had a pro-migratory effect on the keratinocytes, with the best results obtained for KSLW, DAL-PEG-KSLW and CAR. Migration of the cells in this case was comparable to the effect induced by 10% FCS and more pronounced than in case of the control probe. It is worth of mentioning, that migration stimulated by the conjugate DAL-PEG-KSLW (Fig 3) was about 15% higher than that observed for the native peptides KSLW and DAL.

pone.0140377.g002: Effect of the peptides on HaCaT keratinocytes and fibroblasts cell migration in the in vitro scratch test.HaCaT cells and fibroblasts were grown in 6-well plates until the 100% confluence was reached, then cells were starved for the next 12 hours in DMEM without 10% FCS and scratched once vertically with a 200μL pipette tip. After being washed two times with sterile PBS, a fresh portion of DMEM medium was added and cells were treated with the tested peptides applied at their optimal doses (25 μg/mL). Migration was analyzed after 24 hours by means of Zeiss Observer D1 microscope, wounding areas were analyzed with AxioVision software and expressed as the percentage of the wound width in comparison to the control sample (cells incubated in DMEM without FCS). Control+ FCS corresponds to the sample with cells incubated in the medium containing 10% FCS and was treated as the additional positive control. Data represent the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control.

Mentions:
As illustrated in Fig 2 all of the tested peptides had a pro-migratory effect on the keratinocytes, with the best results obtained for KSLW, DAL-PEG-KSLW and CAR. Migration of the cells in this case was comparable to the effect induced by 10% FCS and more pronounced than in case of the control probe. It is worth of mentioning, that migration stimulated by the conjugate DAL-PEG-KSLW (Fig 3) was about 15% higher than that observed for the native peptides KSLW and DAL.

Bottom Line:
Herein, we present the initial results of our studies aimed at the design of bifunctional peptide-based compounds.The chemical approach, that was utilized in this work, was based on the conjugation of antimicrobial peptides with the peptides, that have potential pro-proliferative and/or cytoprotective activity towards human keratinocytes and fibroblasts, in order to obtain antimicrobials with reduced cytotoxicity or compounds that maintain both activities, i.e. inhibit bacterial or fungi growth and activate cell proliferation/migration in in vitro tests.As a result, we obtained a group of peptide conjugates that effectively inhibited the growth of selected bacterial and fungi strains and were able to stimulate proliferation and migration of keratinocytes and fibroblasts under their effective microbicidal concentrations.

Affiliation:
Department of Biochemistry, Faculty of Chemistry, University of Gdansk, Gdansk, Poland.

ABSTRACTSkin represents the largest organ of the human body and plays a crucial role in its protection from the negative impact of the outside environment, maintains its homeostasis, enables sensory interaction and thermoregulation. The traumatized skin tissue undergoes several phenotype switches due to progressive reoxygenation and release of cytokine and growth factors, that activate mechanisms of reparative processes. However, in case of wounds colonized with pathogenic microflora natural regenerative mechanisms become substantially impaired, that could lead to chronic inflammatory states with non-healing skin lesions. Herein, we present the initial results of our studies aimed at the design of bifunctional peptide-based compounds. The chemical approach, that was utilized in this work, was based on the conjugation of antimicrobial peptides with the peptides, that have potential pro-proliferative and/or cytoprotective activity towards human keratinocytes and fibroblasts, in order to obtain antimicrobials with reduced cytotoxicity or compounds that maintain both activities, i.e. inhibit bacterial or fungi growth and activate cell proliferation/migration in in vitro tests. As a result, we obtained a group of peptide conjugates that effectively inhibited the growth of selected bacterial and fungi strains and were able to stimulate proliferation and migration of keratinocytes and fibroblasts under their effective microbicidal concentrations.